Glycopolymers are major components of the extracellular matrix of organisms. These matrixes can be remodeled and/or degraded by endoglycosidases and exoglycosidases. Examples of endoglycosidases are chitinase, lysozyme, heparanase, hyaluronidase, and cellulase. Chitinase.
Chitin is the linear polymer of beta 1-4 linked N-acetylglucosamine residues. It is the second most abundant glycopolymer on earth that is present in cell walls and coatings of a very large variety of organisms. Chitin is degraded in a stepwise manner by the concerted action of endoglycosaminidases (chitinases) and exoglucosaminases. The chitinases are widely distributed in nature and are known to fulfill several critical biological functions. Examples are roles in food processing, remodeling and defense against chitin-containing pathogens. Chitinases can be also industrially employed, for example in crop protection, food preservation, bio-degradation of chitin-containing waste product and production of chito-oligomers or other fine chemicals. A novel area of application is in the field of diagnosis and monitoring of specific human disease conditions as well as the assessment of chitinase deficiency as potential risk factor for specific infections.
The existence of endogenous chitinases in man has only recently been unequivocally demonstrated (Hollak et al., 1994). The enzyme was initially discovered in patients with Gaucher disease, an inherited lysosomal storage disorder that is caused by a deficiency in the lysosomal enzyme glucocerebrosidase. The level of chitinase activity is generally 1000-fold increased in plasma of symptomatic Gaucher patients. The corresponding enzyme, characterized in great detail at the level of protein, RNA and gene, has been named chitotriosidase. The features of the enzyme have been described in a series of publications (Renkema et al., 1995; Boot et al., 1995; Renkema et al., 1997; Renkema et al., 1998; Boot et al., 1998; Boot et al., 2001). It was shown by us that in tissues of Gaucher patients, macrophages transform to lipid-laden pathological cells that synthesize and secrete large quantities of chitotriosidase. The concomitant marked elevation of enzyme in plasma reflects the presence of abnormal macrophages in tissues and is currently used for the diagnosis, the detection of onset and progression of disease and the monitoring of efficacy of therapeutic intervention (Aerts et al., 1997; Cox et al., 2000; Casal et al., 2002, Giraldo et al., 2002). Deficiency in the enzyme is a frequent trait and is predominantly caused by a 24 bp duplication in the chitotriosidase gene (locus 1q31). In various ethnic groups the frequency of carriers for this mutation is about 35%. A convenient test for establishing an individual's chitotriosidase genotype has been developed (Boot et al., 1998; Choi et al., 2001).
Chitotriosidase is the true analogue of chitinases from lower organisms. It can efficiently degrade chitin, releasing chitotriose and chitobiose fragments from the reducing end of the polymer. Recently, a human mucinase was discovered which also displays chitinase activity (Boot et al., 2001). The enzyme shows an extreme acid stability and acid pH optimum for activity. This enzyme has been named acidic mammalian chitinase (AMCase). It is not present in serum in contrast to chitotriosidase.
It has been earlier conceived that expression of chitinase activity is a very specific phenomenon that is uniquely related to chronic activation of tissue macrophages. This novel thought might be exploited for various diagnostic purposes and could also revolutionize non-invasive monitoring of progression of diseases and correction by therapies. Proof of concept has indeed been obtained: chitotriosidase has been found to be expressed by chronically activated macrophages in several pathological conditions resulting in elevated enzyme activity levels in bodily fluids such as plasma, cerebral spine fluid or urine. Examples of conditions showing such type of abnormalities are several lysosomal lipid storage disorders (Guo et al., 1995) sarcoidosis and visceral Leishmaniasis (Hollak et al, 1994), thalassemia (Barone et al., 2001), arteriosclerosis (Boot et al, 1999), HES (hydroxyethylstarch)-induced prunitis, CGD (chronic granulomatosis disease), Crohn's disease, Tangier disease, and arteriosclerosis (Aerts and coworkers, not yet published observations).
Detection and quantitation of chitotriosidase protein in plasma with immunological techniques is problematic. In healthy subjects the protein concentration is on average only 5 ng/ml of serum.
The fact that the protein can catalyse a specific reaction allows in principle a more sensitive and quantitative detection by measuring this enzymatic activity. Unfortunately, again several limitations exist in this connection. Chitin degradation is generally followed using colloidal chitin as substrate and the indirect detection of released fragments. The latter is accomplished by hydrolysis of fragments upon incubation with hexosaminidase, whereafter free N-acetylglucosamine moieties are detected. This method is very insensitive and not directly proportional to the initial chitinase activity. Assays using as substrate radiolabeled chitin or chitins conjugated with chromophores have also a very poor sensitivity and do not render reliable results with plasma samples. A several hundred-fold more sensitive assay has been earlier described by us (Hollak et al.,1994). The assay is based on the use of 4-methylumbelliferyl-chitotrioside as substrate. Alternatively, 4-MU-chitobiose or 4-MU-chitotetraose can be used as substrate (Hollak et al., 1994). Chitotriosidase is able to cleave these synthetic substrates, thus releasing the fluorescent 4-methylumbelliferone. The assay with the above mentioned 4-MU-substrates has a major drawback. No use can be made of saturating substrate concentrations due to apparent substrate inhibition (see for example FIG. 1). Consequently, the assay has to be performed at sub-saturating substrate concentration. As inevitable result of this, the measured enzymatic activity is intrinsically not strictly related to the input of enzyme and only linear in time for a very short period. The results of assays with plasma samples can only be well interpreted if additionally parallel assays are run with standard (pure) chitotriosidase preparations and assay time and input of plasma protein are extensively varied. Considerable expertise is required to obtain reproducible results with this method. In conclusion, at present no convenient and widely applicable method is available that allows very sensitive and quantitative detection of chitotriosidase and comparable chitinases.
Lysozyme.
Lysozyme catalyzes the hydrolysis of certain mucopolysaccharides of bacterial cell walls. Specifically, it catalyzes the hydrolysis of the bacterial cell wall beta(1-4) glycosidic linkages between N-acetylmuramic acid and N-acetyiglucosamine, but not the glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid. Lysozyme is involved in bacteriolytic defensive and immune response. It is present in organs and bodily fluids of the human, including plasma, where it is found in monocytes and macrophages, neutrophils and g˜andular cells. Serum lysozyme is a potential marker for activity of monocytes and macrophages. The serum level of lysozyme is for instance elevated in patients with an infection, autoimmune disease and/or cancer. Examples of bacterial and viral infections are tuberculosis, pneumonia, meningitis, otitis media, urinary tract infections, sepsis and acquired immunodeficiency syndrome. Examples of autoimmune diseases are rheumatic disorders as e.g. rheumatoid arthritis, Inflammatory Bowel Disease, as e.g. Ulcerative Colitis and Crohn's disease, chronic respiratory inflammatory diseases as e.g. chronic bronchitis and chronic sinusitis, interstitial pulmonary diseases as sarcoidosis, and diabetes. Examples of cancer are colorectal cancer, leukemia as e.g. Hodgkin's disease and lung carcinoma.
For measurement of the activity of lysozyme several assays are available. Lysozyme is most frequently analyzed by a variety of methods using Micrococcus lysodeikticus cells as substrate, such as the turbidimetric assay. Some improvement in the turbidimetric method was obtained by Morsky (1983). This modified lysozyme assay has improved routine clinical use in regard to analysis speed, sensitivity, linearity and reproducibility. The reaction course of bacterial cell wall lysis for quantifying lysozyme in serum and urine can also be applied to the solid phase with agarose gel as reaction medium: the lysoplate method (Maeda et al., 1980). The reproducibility of this method is reasonable but labour intensive. Furthermore the results are not always reproducible due to biological factors in the sample or agar batch.
In contrast to the turbidimetric method, the enzyme-linked immunosorbent assay (ELISA) (Taylor et al., 1992), the double-antibody radioimmunoassay (Thorsteindottir et al., 1999) and the electroimmunodiffusion technique measure the concentration of lysozyme. Comparison of lysozyme values obtained with the ELISA and turbidimetric methods showed good correlation. The major drawback in both the ELISA, radioimmunoassay and electroimmunodiffision assay is still the fact that neither of these immunological techniques measures the activity of lysozyme. Their use is also limited by their degree of sensitivity and the presence of lysozyme isozymes. Another drawback concerning lysozyme assays is the lack of uniformity in standardization of lysozyme assays. Thus, the results are not always unambiguously interchangeable, and clinical reference values differ greatly. In conclusion, a simple, rapid, sensitive and specific assay to measure the lysozyme activity is not available.
Hyaluronidase
Hyaluronan, hyaluronic acid (HA), is one of the principal glycosaminoglycans of the extracellular matrix. It consists of a high molecular weight polymer of repeating units of N-acetylglucosamine and D-glucuronic acid. It is believed to have numerous important biologic functions, including modulation of cell proliferation, migration and differentiation and is believed to be crucial in tissue remodeling, e.g. during embryogenesis and wound healing, in tumorigenesis, angiogenesis and inflammation.
Clinically, aberrations of HA metabolism are associated with processes such as adult respiratory distress syndrome, organ transplant oedema and rejection, and as a marker for cancer remission and relapse. The binding of exogenous FIA to cell surface receptors, the most important one being CD44, mediates endocytosis of extracellular HA, leading to its degradation by lysosomal hyaluronidase. Hyaluronidase is the enzyme that degrades HA. This endoglycosidase cleaves the N-acetyl-(1-*4)-glucosaminic bonds in HA, forming even-numbered oligosaccharides, with mainly tetrasaccharides as the smallest fragments (Menzel et al., 1998); digestion with testicular hyaluronidase results in the exclusive cleavage of the N-acetylhexosaminidic linkages, the products comprising a series of oligosaccharides with N-acetylglucosamine at the reducing terminus. Upon exhaustive digestion, the tetrasaccharide is the major product, closely followed by the hexasaccharide and smaller amounts of higher oligosaccharides, while only a small proportion of disaccharide is found in a digest of this type.
Hyaluronidases from vertebrate tissues can be separated into two classes that have very different biological functions. The major hyaluronidase in human plasma is the hyaluronoglucosamindase-1. This enzyme is expressed in multiple tissues. In the sperm plasma membrane and acrosomal membrane an enzyme with hyaluronidase activity is present, the sperm adhesion molecule 1 gene or PH-20. To penetrate the cumulus layer, a hyaluronan-rich extracellular matrix surrounding the oocyte, the sperm uses this enzyme to break down the hyaluronan. This enzyme is a marker for the sperm function.
Hyaluronan (HA) and hyaluronidase (HAase) are involved in malignant transformation and cancer progression. In many malignancies, levels of HA correlate with metastatic behaviour while HAase suppress malignant progression. In bladder cancer, hyaluronic acid and hyaluronidase are used as biological markers for bladder tumour, angiogenesis and metastasis, being secreted in urine. An elevated urinary HA indicates the diagnosis of bladder cancer regardless of tumour grade and the urinary HAase levels correlates with the malignant potential of bladder cancer, being grade 2 and grade 3 (Lokeshwar et al., 2000). The hyaluronic acid and hyaluronidase (HA-HAase) test is the only non-invasive assay described that detects HA and HAase, because it measures in urine. Other HA and HAase measuring tests use sera as biological samples. The HA-HAase test for urine is an ELISA-like assay using e.g. an avidin-biotin-peroxidase color detection system. This test has 90-92% sensitivity and 80-84% specificity for bladder cancer (and is currently evaluated in multicenter trial settings (Lokeshwar, 2001).
Despite the sensitivity and specificity of the HA-HAase test, in current practice cystoscopy is the gold standard for detecting bladder cancer and evaluating tumor recurrence, while voided urine or bladder wash cytology is often used as an adjunct to cystoscopy for detecting high-grade bladder cancer. The voided urine cytology is the standard non-invasive tumor specific marker and evaluates malignancy based on cellular morphology. Voided urine cytology has an excellent specificity but a poor sensitivity (Brown 2000). However, it is not sensitive for detecting low-grade disease. Also variability among those interpreting cytology findings is significant. Moreover, urine cytology is not quantitative. In conclusion, urine cytology in its present form cannot replace cystoscopy as a method for detecting and monitoring bladder cancer, but should be used as an additional application. An ideal non-invasive test should be sensitive, specific, rapid, technically simple and have low intra-assay and interassay variability.
The most commonly used hyaluronidase assays, which are either insensitive or lack specificity, are based upon the measurement of the generation of new reducing N-acetylamino groups, or loss of viscosity or turbidity. Other assays are dye binding assays, zymogram electrophoretic technique or enzyme activity measurement based on the Morgan-Elson reaction. All these assays are relatively cumbersome and insensitive. For large-scale measurements application of the modern ELISA-like microtiter assay by Frost and Stem (1997) may be more convenient. This study describes a sensitive, rapid microtiter-based assay for hyaluronidase activity that does not require highly specialized biological reagents. However, this assay is not accurate, time- and dose-dependent. Hyaluronidase activity can also be measured in venoms using capillary electrophoresis or by USP XXII assay but also these assays are not sensitive as described by Pattanaargson et al., 1996.
Heparanase.
Heparan sulfate (HS) and heparan sulfate proteoglycans (HSPGs) are acidic complex polysaccharides found on the cell surface, in the extracellular matrix and vascular basal lamina. These biopolymers play an important role in cell proliferation, differentiation, migration and shape. The HS chains are originally synthesized as a polysaccharide of alternating N-acetyl-glucosamine (GlcNAc) and glucuronic acid (GlcUAu) that are enzymatically modified to a complex polysaccharide containing sulphate rich and sulphate poor regions. The enzyme heparanase (Hpa) cleaves the heparan sulfate glycosaminoglycans from the proteoglycan core proteins and degrade them to small oligosaccharides. Heparanase is an endoglucuronidase, cleaving the linkage between glucuronic acid (GIcUA) and N-acetyl-glucosamine (GlcNAc). Hpa I, the dominant heparanase in mammalians, is a hydrolase cleaving the HS chains at specific places and not an eliminase as heparinase from Flavobacterium heparinuin. Heparanase is expressed in a wide variety of tissues and cells. (Barne, 2001 and Parish et al, 2001). It plays a major role in early embryogenesis, morphogenesis, pregnancy and development to inflammation, wound healing, and tumor angiogenesis and metastasis. Human platelets have been shown to contain high levels of heparanase activity, capable of degrading endothelial cell surface, tumour-derived and ECMderived HSPG as well as free HS chains and heparin. Heparanase is able to facilitate cell invasion by degrading the extracellular matrix and the vascular basement, needed for invasion of cancer in the metastatic phase and neovascularization. (See for review Parish et al, 1998 and Vlodavsky et al, 2001). The metastatic potential of tumour cells is related to their increased heparanase content. A heparanase assay is a diagnostic tool for cancer staging, since it has been suggested that heparanase plays a prominent role in cancer-associated processes. Indeed, it has been found that cancer patients had twice the serum heparanase levels as normal healthy adults.
It has been shown that HpaI can cleave the antithrombin-binding site in heparin, low molecular weight heparin (LMWH) and synthetic pentasaccharides containing an intact antithrombin III binding site, thereby inhibiting the anticoagulation function of these drugs that are given therapeutically or prophylaxis to patients with an increased risk for thrombosis. Heparin, LMWH and synthetic pentasaccharides are indicated as therapeutical in the acute phase of deep venous thrombosis or pulmonary embolism treatment and as prophylaxis for e.g. general surgery, acute myocardial infarction, ischemic stroke, intensive care patients and bedridden patients with a risk factor.
During therapy of patients with heparin, LMWH or pentasaccharides it is essential to monitor the activated clotting time to guide the heparin therapy during cardiac surgery, cardiac catherication and coronary interventions. The available clotting assays to measure the coagulation status of patients in heparin therapy are sensitive to the presence of small traces of heparin and tissue factor pathway inhibitor (Bladbj erg et al., 2000) and the presence of the endogenous heparanase in plasma. A simple heparanase/heparinase assay would be medical useful to monitor the therapy of the patients with heparin, LWMHs or synthetic polysaccharides and scientific useful to elucidate the pathways.
It has been shown that synthetic heparin mimicking compounds can inhibit heparanase and decrease the incidence of metastases. The role of heparanase in cancer and angiogenesis has not been elucidated, although the heparanase activity has been known for several decades. A major reason for the lack of studies of heparanase activity and heparanase inhibition has been due to the absence of a simple, rapid and sensitive assay for heparanase activity (Parish et al., 1999). Also for monitoring the heparanase activity in cancer and during therapy of patients with anticoagulants a reliable assay is necessary. In addition, a simple assay to measure heparanase activity would also be helpful in studying the activity of homologous proteins as e.g. the mammalian gene Heparanase 2 (McKenzie et al., 2000).
Different kinds of assays have been described for measuring heparanase activity. In one type of assay heparanase activity is detected by separating cleaved from uncleaved radioactivity labelled and fluorescence labelled HS using electrophoresis. In another type of assay heparanase activity is detected by incubating the sample in the presence of a solid phase support having immobilized thereon a substrate for the heparanase and separating cleaved from uncleaved substrate. Freeman et al. (2001) describe an example for this type of assay using histidine-rich glycoprotein as heparan sulphate binding protein. Another example is the assay described by Brenchley (2000), wherein two solid phase supports are used, one to bind the uncleaved and one to bind the cleaved HS. These assays are laborious. A simple, rapid, sensitive and specific assay to measure the heparanase activity is not available.
Cellulase.
Cellulose, the most abundant carbohydrate produced by plants, is an unbranched (1-4)-˜-D-glucose polymer with repeating unit of cellobiose (glucose dimer) instead of glucose. Although cellulose is a simple polymer, it forms insoluble, crystalline microfibrils, but it also contains regions with less structure, the so-called amorphous zones. All organisms known to degrade cellulose efficiently produce a battery of enzymes with different specialties, which act together in synergism. This enzymatic hydrolytic system consists of three different enzymes and so catalytic reactions: (i) endoglucanases (EC), which can randomly hydrolyse the 1,4-˜-glycosidyl linkages within the water-insoluble cellulose chains; (ii) exoglucanase or cellobiohydrolases (CBHs), which hydrolyse the 1,4-˜-glycosidyl linkages of either the reducing or non-reducing ends of cellulose chains to form cellobiose and (iii) ˜3-glucosidases or cellobiose, which converts the water soluble cellobiose into two glucose residues. Microorganisms, especially fungi, produce often mixtures of these enzymes. Together, these enzymes form a cellulolytic system and hydrolyse insoluble cellulose, both crystalline and amorphous, in a very efficient and synergistic way.
Several applications of cellulases are being developed for textile, food, and paper pulp processing (Beguin et al, 1994). Today, cellulase enzymes are used in different segments of the cotton textile industry. For the characterization of the cellulase in these contexts and for the insight in the complex mechanisms of hydrolysis, a rapid, sensitive test is desired.
Glucanase.
Beta-Glucans with 1,3-˜3-glycosidyl linkages are present in a variety of organisms. Various beta 1,3 glucanases that cleave 1,3-˜3-glycosidyl linkages have been described in lower organisms and plants. The cell wall of various pathogenic organisms contains beta 1,3-glucan and this structure exerts a potent effect on the immune system in man. Nothing is so far known about the catabolism of beta 1,3 glucan in man. Accurate detection of beta 1,3-glucanase activity is therefore highly desired.
Thus, endoglucanases are involved in a wide range of important (patho)biological processes. The concentration of such enzymes is often altered during a disease and during the process of counteracting/curing of a disease. Hence, the concentration of a certain endoglucanase is often indicative for the status of an individual. Detection of the concentration of such enzymes provides important information about a disease and/or the treatment of a disease. However, in spite of different tests developed in the art, detection of these enzymes is still cumbersome. The aim of the present invention is to provide improved, sensible and simple methods for detecting an activity of an endoglycosidase.